A. Summary Aging is a major risk factor for degenerative diseases, and associated with cumulative protein damage and the decline of cellular function. Our previous work demonstrated that the expression of aggregation-prone proteins in C. elegans models of neurodegenerative disease initiates a cascade of protein damage that results in misfolding of other metastable proteins. Using a computation approach, we showed that proteins at-risk for aggregation are not randonri, but rather have in common sequence elements that predict their intrinsic metastability and tendency to aggregate. Moreover, misfolding and aggregation in various neuronal and nonneuronal tissues is not sporadic throughout lifespan, but rather occurs at a much earlier point in C. elegans adulthood coincident with a dramatic decline in the robustness of inducible cell stress responses. In this MERIT extension application, we propose to test the hypothesis that the composition of the proteome. and alterations in the balance of soluble and insoluble species during aging are accelerated by acute and chronic proteotoxic stress and protected by selective induction of proteostasis network and lifespan pathways. This will be demonstrated using two complementary model systems: an organismal approach using C. elegans that affords a precise model for aging and the ability to test genetic pathways that control proteostasis and lifespan, .and adult human primary cells and inducible pluripotent cells to assess whether changes in the aging proteome and mechanisms that control these processes are conserved. The Aims are: (1) Assessing the specificity and selectivity of proteome aggregation in acute and chronic proteotoxic stress and aging. We will perform a proteomic analysis at multiple points of C. elegans adulthood and aging, and quantify the soluble and aggregated fractions in animals challenged by acute (heat shock) and chronic (expression of polyglutamine. A, and tau) proteotoxic stress. Proteins that shift to the aggregated state will be validated by generating a corresponding series of transgenic protein-GFP reporter lines and used to assess folding transitions in different compartments and tissues in living animals. Comparative proteomic analysis of human primary cells from donors over a wide range of chronological age will reveal whether the same proteins or pathways undergo similar changes in solubility and aggregation. The proteomic data will be integrated with a corresponding set of RNA-seq data and used to develop models to establish whether proteomic risk, failure, or protection can be assessed at the tissue-level to establish an organismal understanding of proteome-wide networks. (2) Establishing whether proteome stability can be selectively altered by regulation of the proteostasis networif and lifespan pathways. We will determine the consequences to the soluble and aggregated proteome of enhancing or inhibiting different arms of the PN. for example by constitutive activation or inhibition of the heat shock response and the organellar unfolded protein responses. Likewise, are the consequences to the proteome the same or distinct, by activating or inhibiting the lifespan pathways regulated by caloric restriction, germline stem cell signaling, insulin-signaling, and the heat shock response, and '(3) Developing a multi-dimensional systems network map of the stressed and aging proteome. To develop a visual image of proteome dynamics over chronological age using data from Aims 1 and 2. together with archival data on expression of the proteome and the PN. This map will be used to predict the proteins, compartments, or tissues that are affected by aging and stress, and protected by chaperone networks and the PN.